In recent years solid phase biochemistry (e.g. Solid Phase Biochemistry--analytical and synthetic aspects, W. H. Scouten, editor, John Wiley & Sons, New York, 1983) has found wide application in biotechnology. Major interests have focussed on affinity chromatography (e.g. Affinity Chromatography--a practical approach, editors P. D. G. Dean, W. S. Johnson & F. A. Middle, IRL Press Ltd., Oxford, 1986), Nucleic Acid Hybridization--a practical approach, editors B. D. Hames & S. J. Higgins, IRL Press Ltd., Oxford, 1987), immobilized enzymes and cells (e.g. Immobilized Cells and Enzymes--a practical approach, editor J. Woodward, IRL Press, Oxford, 1985), solid phase peptide (e.g. G. Barany & R. B. Merrifield in `The Peptides`, Vol. 2; editors: E. Gross & J. Meienhofer, Academic Press, New York, 1979) and oligonucleotide synthesis (e.g. Oligonucleotide Synthesis--a practical approach, editor M. J. Gait, IRL Press Ltd., Oxford, 1984). In almost all cases as given in the references cited above the nucleic acids or peptides/proteins are either adsorbed or non-specifically linked to beaded material such as cellulose, glass beads, Sephadex, Sepharose, agarose, polyacrylamide, porous particulate alumina, hydroxyalkyl methacrylate gels, diol-bonded silica or porous ceramics. Flat material such as filter disc of nylon and nitrocellulose are very frequently used to immobilize nucleic acids for hybridization experiments by adsorption. In some applications in this area chemically modified paper is employed; cellulose is either functionalized with a diazobenzyloxymethyl (J. C. Alwine et al. in Methods in Enzymology, Vol. 68, editor: R. Wu, Academic Press, New York and London, page 220, 1979) or a O-aminophenylthioether (B. Seed, Nucleic Acids Res. Vol. 10, page 1799, 1982) derivative, which in both cases leads to a non-specific covalent linkage of nucleic acids to the paper. In another attempt the surface of tubes made from vinylacetate-ethylene copolymers was chemically activated to furnish a non-specific covalent attachment of proteins to the tube surface (G. Manecke & H. G. Vogt, J. Solid-phase Biochem., vol. 4, page 233, 1979). It should be noted that in the latter case no porous structure is available to supply a significant amount of molecules to be attached to the carrier.
Recent attention has focussed on the development of methods for the site specific covalent attachment of biomolecules to solid supports. Synthetic DNA molecules covalently bound to bead matrices such as cellulose (P. T. Gilham in Methods in Enzymology, editors L. Grossman & K. Moldave, vol. 21, part D, page 191, Academic Press, New York and London, 1971 and J. T. Kodanaga & R. Tjian, Proc. Natl. Acad. Sci. USA, Vol. 83, page 5889, 1986), glass beads of controlled porosity (T. Mizutani & Y. Tachibana, J. Chromatogr., Vol. 356, page 202, 1986) and latex microspheres (J. N. Kremsky et al. Nucleic Acids Res. Vol. 15, page 2891, 1987) have been used for affinity purification of complementary nucleic acids and for sequence specific binding of proteins and as reactants in enzymatic ligation reactions. Likewise synthetic peptides attached to various beaded carriers, including sepharaose and agarose, have been widely used for affinity isolation of enzymes (P. Cuatrecasas, M. Wilchek & C. B. Anfinsen, Proc. Natl. Acad. Sci. USA, vol. 61 page 636, 1968), antibodies (E. Hurwitz et al., Eur. J. Biochem., vol. 17, page 273, 1970) and other proteins (B. Penke et al., J. Chromatogr., Vol. 376, page 307, 1986).
Synthesis of affinity matrices usually involves the reaction of a support bound electrophilic function with a nucleophilic group within the oligonucleotide or within the peptide. Conversely, the electrophilic function may be on the biomolecule and undergoes reaction with a nucleophilic group on the polymeric support.
More often, peptides are coupled to solid carriers via the various reactive functional groups of the amino acid side chains as well as through the amino and carboxyl termini of the biopolymer. Oligonucleotides are relatively more difficult to attach to solid supports because they do not contain any strong nucleophilic or electrophilic centers. As a result, a number of methods and reagent have been described that allow for the chemical synthesis of oligomers containing reactive functionalities at defined positions in the molecule, preferentially at one of the termini of the biopolymer (see, e.g. J. M. Coull et al., Tetrahedron Lett. vol. 27 page 3991, 1986; S. Agrawal et al., Nucleic Acids Res vol. 14, page 6227, 1986; B. A. Conolly, Nucleic Acids Res., vol. 15, page 3131, 1987; B. A. Conolly and P. Rider, Nucleic Acids Res., vol. 12, page 4485, 1985).
Since both approaches require the synthesis and isolation of an oligonucleotide or peptide prior to attachment to the solid matrix, a significant improvement would be the direct solid phase synthesis of the biomolecule onto the support. In this way the affinity support can be directly generated. Two prior examples of this approach include the chemical synthesis of oligo-dT on cellulose beads (P. T. Gilham, see above) for the affinity isolation of poly A tail containing mRNA and the synthesis of short peptides on polyethylene pegs useful for antibody epitope mapping by employing the specific affinities of certain amino acid sequences on the antibody to react strongly and specifically with the antigen (H. M. Geysen et al., Proc Nat'l. Acad. Sci. USA, vol. 82, page 3998, (1984)). Polyethylene pegs are only useful for very specific purposes and suffer from the extreme low loading of immobilized biomolecules due to the non-porous structure. In a description of a process for the simultaneous chemical synthesis of several oligonucleotides paper discs have been used (DE No. 3301833 and EP No. 114599). This material cannot be recommended to serve as affinity support, because the material apparently does not allow to use the state-of-the-art phosphoamidite chemistry for the construction of long oligonucleotides with more than one hundred nucleotide units in the sequence (N. D. Sinha et al., Nucleic Acids Res., 12:4539, (1984)). With the phosphate triester method (see e.g. M. Gait as cited above) only relatively short oligonucleotides (in the range of twenty nucleotide units containing sequences) can be obtained with the paper disc method. Moreover, after a few synthetic cycles employing the necessary treatment with different reagents and washing steps the paper gets very fragile and looses its mechanical stability. No peptides have been synthesized so far on paper; it is very probable that due to the harsh conditions necessary to synthesize peptides the cellulose matrix will be disrupted. Thus, affinity supports cannot be obtained by virtue of chemical synthesis of oligonucleotides or peptides onto paper as solid support.
Nucleic acids and peptides or proteins have been immobilized onto beaded and flat polymeric supports either by adsorption or by non-specific covalent linkage. To mediate an efficient and specific interaction using hybridization or affinity techniques between the soluble and immobilized biomolecules, a specific covalent attachment of the biomolecule involving only one terminal function would be optimal. This would make available the whole sequence of the immobilzed biomolecule to interact with the complementary molecule in solution. Adsorption or non-specific covalent binding, however, involves several functions in the biomolecule, which are then rendered unavailable for the desired intermolecular interaction. Adsorption has furthermore the disadvantage that some of the immobilized biomolecules can be washed out (desorbed) during the hybridization or affinity process. This has to be particularly considered if the affinity support should be reused several times.
Whereas the terminus specific covalent attachment of oligonucleotides or peptides onto solid supports using the stepwise synthetic approach has been performed using beaded supports or paper discs (in the case of oligonucleotides) or beaded supports and polyethylene pegs (in the case of oligopeptides) no synthesis of these biopolymers has been reported employing membrane-type supports.
A membrane, a being flat and highly porous, mechanical stable material, would be most advantageous as affinity support, because it could be handled easily, cut into various sizes, stacked on top of each other for upscaling purposes and reused several times. Furthermore, the support should be chemically stable under the conditions of oligonucleotide and peptide synthesis and should not show non-specific binding of either nucleic acids or proteins as this would give rise to a sensitivity-reducing background interaction. The development of an affinity support which fulfills these different requirements is not a trivial task. Whether the direct chemical synthesis of oligonucleotides or peptides is possible on such an insoluble support can also not be predicted. As mentioned, paper could only serve as a support for solid phase oligonucleotide synthesis when the phosphotriester approach was employed; for reasons which are still unclear, the much more efficient and state-of-the-art phosphoamidite chemistry which is very successfully used on porous glass beads did not work on paper.